Improved Chemical-Genetic Fluorescent Markers for Live Cell Microscopy

Area of Science:

• Molecular Biology
• Biotechnology
• Cellular Imaging

Background:

• Inducible chemical-genetic fluorescent markers are crucial for live cell imaging, demanding high spatiotemporal resolution and low background fluorescence.
• The fluorescence-activating and absorption shifting tag (FAST) system utilizes synthetic fluorogenic chromophores for fluorescent labeling.

Purpose of the Study:

• To rationally design and engineer improved variants of the FAST protein with enhanced fluorescence performance.
• To evaluate the impact of protein modifications on binding affinity and fluorescence quantum yield with various fluorogens.

Main Methods:

• Rational design was employed to modify the protein binding pocket of the FAST system.
• Screening of modified proteins was performed with four different synthetic fluorogens to assess fluorescence properties.
• A single point mutation was introduced, leading to the development of improved FAST (iFAST).

Main Results:

• A single mutation in the FAST protein significantly improved both quantum yield and dissociation constant across most tested fluorogens.
• The engineered iFAST protein enabled the creation of tandem reporters (td-iFAST) exhibiting enhanced brightness compared to standard fluorescent proteins.
• td-iFAST generated green and red fluorescent reporters that were 1.6-fold and 2-fold brighter than EGFP and mCherry, respectively, at a comparable size.

Conclusions:

• The iFAST system represents a significant advancement in chemical-genetic fluorescent markers, offering superior brightness and efficiency for live cell imaging.
• Engineered FAST variants provide versatile and brighter fluorescent reporters, expanding possibilities for high-resolution cellular studies and diagnostics.